Rotavirus outer capsid protein VP4 is a major independent protective antigen. A cDNA clone representing the VP4 gene of symptomatic human rotavirus strain KU (VP7 serotype 1) or DS-1 (VP7 serotype 2) or asymptomatic human rotavirus strain 1076 (VP7 serotype 2) was constructed and inserted into a baculovirus expression vector under the control of the polyhedrin promoter. The resulting recombinants expressed the appropriate authentic VP4 protein. Guinea pigs immunized with these VP4 proteins developed specific antibodies which neutralized infectivity of the rotavirus from which the immunizing VP4 was derived. These antisera were then used in neutralization tests to define the extent and distribution of VP4 antigenic polymorphism among human rotaviruses. Three distinct serotypes and one subtype of the VP4 outer capsid protein were identified among 17 human rotavirus strains that had previously been assigned to 5 distinct VP7 serotypes. For the most part, VP4 serotype segregated independently of VP7 serotype. Among the 10 rotavirus strains whose VP4 gene was previously sequenced, there was complete concordance between assignment of VP4 serotype by neutralization and classification according to VP4 amino acid homology. Thus, rotaviruses that exhibited a VP4 amino acid homology of 89% or greater belonged to the same VP4 serotype as determined by neutralization. Finally, evidence was obtained that the serotype-specific domain is located on the VP8 cleavage subunit of VP4.